Adenine nucleotide metabolism in relation to purine enzymes in liver, erythrocytes and cultured fibroblasts.

To evaluate the regulation of adenine nucleotide metabolism in relation to purine enzyme activities in rat liver, human erythrocytes and cultured human skin fibroblasts, rapid and sensitive assays for the purine enzymes, adenosine deaminase (EC 2.5.4.4), adenosine kinase (EC 2.7.1.20), hyposanthine phosphoribosyltransferase (EC 2.4.28), adenine phosphoribosyltransferase (EC 2.4.2.7) and 5'-nucleotidase (EC 3.1.3.5) were standardized for these tissues. Adenosine deaminase was assayed by measuring the formation of product, inosine (plus traces of hypoxanthine), isolated chromatographically with 95% recovery of inosine. The other enzymes were assayed by isolating the labelled product or substrate nucleotides as lanthanum salts. Fibroblast enzymes were assayed using thin-layer chromatographic procedures because the high levels of 5'-nucleotidase present in this tissue interferred with the formation of LaCl3 salts. The lanthanum and the thin-layer chromatographic methods agreed within 10%. Liver cell sap had the highest activities of all purine enzymes except for 5'-nucleotidase and adenosine deaminase which were highest in fibroblasts. Erythrocytes had lowest activities of all except for hypoxanthine phosphoribosyltransferase which was intermediate between the liver and fibroblasts. Erhthrocytes were devoid of 5'-nucleotidase activity. Hepatic adenosine kinase activity was thought to control the rate of loss of adenine nucleotides in the tissue. Erythrocytes had excellent purine salvage capacity, but due to the relatively low activity of adenosine deaminase, deamination might be rate limiting in the formation of guanine nucleotides. Fibroblasts, with high levels of 5'-nucleotidase, have the potential to catabolize adenine nucleotides beyond the control od adenosine kinase. The purine salvage capacity in the three tissues was erythrocyte greater than liver greater than fibroblasts. Based on purine enzyme activities, erythrocytes offer a unique system to study adenine salvage; fibroblasts to study adenine degradation; and liver to study both salvage and degradation.     

NUCLEOTIDE METABOLISM IN RAT BRAIN

Abstract— The uptake, the conversion to nucleotides, and their incorporation into RNA for labelled glycine, aspartate, the free bases and nucleosides of purines and pyrimidines were investigated with cortical slices of rat cerebrum. At the end of a 1-hr incubation time the slice-to-medium ratio of the radioactivities for labelled aspartate, glycine, adenine and adenosine were 34, 26, 20 and 5, respectively, while the slice-to-medium ratios for hypoxanthine, inosine, guanine, guanosine, xanthine, orotate, cytidine, cytosine, uridine, and uracil ranged from 1.3:1 to 2:1. Over 99 per cent of the total radioactivity taken up by the cortical slices was present in the TCA supernatant and 86, 82, 65, 50, 34, 23, 20 and 1.6 per cent of this radioactivity was in the form of nucleotides at the end of a 1-hr incubation with labelled adenine, adenosine, hypoxanthine, inosine, uridine, orotate, cytidine, and glycine, respectively. The incorporation of various radioactive precursors into RNA of cortical slices suggests that nucleotides originating from either de novo synthesis or preformed purine derivatives enter the same nucleotide pool utilized for RNA synthesis. The supernatant fraction from homogenized cerebrum was investigated for the presence of various anabolic and catabolic enzymes associated with nucleotide metabolism. These results were correlated with the data from the RNA incorporation studies, and a possible role for AMP: pyrophosphate phosphoribosyltransferase (adenine phosphoribosyltransferase, I.U.B. 2.4.2.7) to achieve intercellular transfer of AMP is discussed.     

A coupled optical assay for adenine phosphoribosyltransferase and its extension for the spectrophotometric and radioenzymatic determination of 5-phosphoribosyl-1-pyrophosphate in mixtures and in tissue extracts

A reliable assay was developed to characterize crude cell homogenates with regard to their adenine phosphoribosyltransferase activities. The 5-phosphoribosyl-1-pyrophosphate (PRPP)-dependent formation of AMP from adenine is followed spectrophotometrically at 265 nm by coupling it with the following two-stage enzymatic conversion: AMP + H2O----adenosine + Pi (5'-nucleotidase); adenosine + H2O----inosine + NH3 (adenosine deaminase). The same principle was applied to develop a spectrophotometric and a radioenzymatic assay for PRPP. The basis of the spectrophotometric assay is the absorbance change at 265 nm associated with the enzymatic conversion of PRPP into inosine, catalyzed by the sequential action of partially purified adenine phosphoribosyltransferase, commercial 5'-nucleotidase, and commercial adenosine deaminase, in the presence of excess adenine. In the radiochemical assay PRPP is quantitatively converted into [14C]inosine via the same combined reaction. Tissue extracts are incubated with excess [14C]adenine. The radioactivity of inosine, separated by a thin-layer chromatographic system, is a measure of PRPP present in tissue extracts. The radioenzymatic assay is at least as sensitive as other methods based on the use of adenine phosphoribosyltransferase. However, it overcomes the reversibility of the reaction and the need to use transferase preparations free of any phosphatase and adenosine deaminase activities.     

Developmental changes in purine phosphoribosyltransferases in human and rat tissues.

1. The hypoxanthine/guanine and adenine phosphoribosyltransferase activities in a wide variety of human tissues were studied during their growth and development from foetal life onward. A wide range of activities develop after birth, with especially high values in the central nervous system and testes. 2. Postnatal development of hypoxanthine/guanine phosphoribosyltransferase was also defined in the rat. Although there were increases in the central nervous system and testes, there was also a rise in activity in the liver, which was less marked in man. 3. A sensitive radiochemical assay method, using dTTP to inhibit 5'-nucleotidase activity, suitable for tissue extracts, was developed. 4. No definite evidence of the existence of tissue-specific isoenzymes of hypoxanthine/guanine or adenine phosphoribosyltransferase was found. Hypoxanthine/guanine phosphoribosyltransferase in testes, however, had a significantly different thermal-denaturation rate constant. 5. The findings are discussed in an attempt to relate activity of hypoxanthine/guanine phosphoribosyltransferase to biological function. Growth as well as some developmental changes appear to be related to increase in the activity of this enzyme.     

Purine-phosphoribosyltransferase activities in rat and mouse tissues and in Ehrlich ascites-tumour cells

1. The activities of adenine phosphoribosyltransferase and hypoxanthine phosphoribosyltransferase in extracts of rat and mouse liver, brain, spleen, heart and kidney, of rat bone marrow and of Ehrlich ascites-tumour cells have been measured. The specific activity of adenine phosphoribosyltransferase in tumour cells (3mμ-moles of nucleotide formed/min./mg. of protein) was between 15 and 60 times the activity of any mouse tissue examined. Hypoxanthine-phosphoribosyltransferase activity was greater in extracts of tumour cells than in mouse tissues, but the difference was not great. 2. The specific activities of both adenine phosphoribosyltransferase and hypoxanthine phosphoribosyltransferase in ascites-tumour cells decreased respectively by 57% and 36% 2 days after the cells had been inoculated into mice. After 4 days of tumour growth the specific activities of both enzymes increased, reaching a maximum at 10 days. 3. The activity of adenine phosphoribosyltransferase in rat-liver extracts increased steadily up to 4 days after partial hepatectomy, and was still above the control value after 14 days. Hypoxanthine-phosphoribosyltransferase activity in extracts of regenerating rat liver did not increase until the second day after operation and had begun to decrease by the fourth day. 4. From the results it was concluded that adenine phosphoribosyltransferase and hypoxanthine phosphoribosyltransferase may be physiologically important enzymes in rapidly growing tissues, and that inhibitors of the former enzyme have potential value in chemotherapy.     

Purine ribonucleotide biosynthesis, interconversion and catabolism in mouse brain in vitro

The relative rates of the synthetic, interconversion and catabolic reactions of purine metabolism in chopped mouse cerebrum were studied. The rates of incorporation of [(14)C]adenine and [(14)C]hypoxanthine into purine ribonucleotides were much less than the potential activities of adenine phosphoribosyltransferase and hypoxanthine phosphoribosyltransferase, and the rates of incorporation were stimulated by the addition of guanosine to the incubation mixture. The availability of ribose phosphates may be a limiting factor for the formation of ribonucleotides from purine bases. The rate of incorporation of [(14)C]adenosine into purine ribonucleotides was at least seven- to eight-fold higher than that of adenine. The radioactivity in adenine ribonucleotides synthesized from adenine and hypoxanthine was about 100- and ten-fold respectively higher than that in the radioactive guanine ribonucleotides. The conversion of inosinate into guanine ribonucleotides was probably limited by the amount of inosinate available, and the conversion of adenine ribonucleotides into guanine ribonucleotides was probably limited by the activity of adenylate deaminase. The rate of catabolism of [(14)C]adenosine was low in comparison with its rate of utilization for ribonucleotide synthesis. A fraction of the [(14)C]hypoxanthine was catabolized to xanthine and urate. [(14)C]Guanine was completely converted into xanthine, mostly by the guanine deaminase that was released during incubation of chopped mouse cerebrum.     

Changes in the activity of enzymes involved in purine "salvage" and nucleic acid degradation during the growth of Catharanthus roseus cells in suspension culture

Changes during growth in the activity of several enzymes involved in purine "salvage", adenine phosphoribosyltransferase (EC 2.4.2.7), guanine phosphoribosyl-transferase (EC 2.4.2.8), hypoxanthine phosphoribosyltransferase (EC 2.4.2.8) and adenosine kinase (EC 2.7.1.20), the enzymes which catalyze the conversion of nucleoside monophosphate to triphosphate, nucleoside monophosphate kinase (EC 2.7.4.4) and nucleoside diphosphate kinase (EC 2.7.4.6), and several degradation enzymes, deoxyribonucleae(s), ribonuclease(s). phosphatase(s), nucleosidase (EC 3.2.2.1), 3'-nucleotidase (EC 3.1.3.6) and 5'-nucleotidase (EC 3.1.3.5) were examined in cells of Catharanthus roseus (L.) G. Don cultured in suspension. In addition, the incorporation of [8-¹⁴C] adenine, [8-¹⁴C] adenine, [8-¹⁴C]hypoxanthine. [8-¹⁴C] adenosine and [8-¹⁴C]inosine into nucleotides and nucleic acids was also determined using intact cells.
The activities of all purine "salvage" enzymes examined and those of nucleoside monophosphate and diphosphate kinases increased rapidly during the lag phase and decreased during the following cell division and cell expansion phases. The rate of incorporation of adenine, guanine, hypoxanthine, and adenosine into nucleotides and nucleic acids was higher in the lag phase cells than during the following three phases. The highest rate of [8-¹⁴C]inosine incorporation was observed in the stationary phase cells. The activity of all degradation enzymes examined decreased when the stationary phase cells were transferred to a new medium.
These results indicated that the increased activity of purine "salvage" enzymes observed in the lag phase cells may contribute to an active purine "salvage" which is required to initiate a subsequent cell division.     

In vitro protein synthesis during germination and vernalization in winter wheat embryos

Protein synthesis during germination at 24?C and vernalizationat 4?C in winter wheat embryos were investigated with a cell-freesystem. During germination, the capacity for protein synthesisincreased in the early stage between 12 and 36 hr of imbibitionthen declined to a final low level between 48 and 72 hr. Thistransition was due to quantitative changes of the activitiesof ribosomal and supernatant fractions in the early stage andmainly to those of the supernatant fraction in the later stage.During vernalization, the capacity for protein synthesis continuedto decline over 15 to 60 days at 4?C. This transition was dueto the change in activity of the supernatant fraction; the activityof the ribosomal fraction was nearly constant. Electrophoretic analysis of in vitro products indicated thatthe high molecular weight proteins present in 12-hr embryoshad disappeared in 48-hr germinated wheat embryos and that theproducts in 24- and 36-hr embryos were types intermediate betweenthose of 12- and 48-hr embryos. The products in each vernalizedembryo resembled those in 24- and 36-hr germinated embryos.Therefore, it was concluded that the mRNA species for translationchanged during germination and vernalization in winter wheatembryos. (Received January 20, 1977; )     

Developmental Changes in the Activity of Enzymes of Purine Metabolism in Rat Neuronal Cells in Culture and in Whole Brain

The activities (Vmax) of several enzymes of purine nucleotide metabolism were assayed in premature and mature primary rat neuronal cultures and in whole rat brains. In the neuronal cultures, representing 90% pure neurons, maturation (up to 14 days in culture) resulted in an increase in the activities of guanine deaminase (guanase), purine-nucleoside phosphorylase (PNP), IMP 5'-nucleotidase, adenine phosphoribosyltransferase (APRT), and AMP deaminase, but in no change in the activities of hypoxanthine-guanine phosphoribosyltransferase (HGPRT), adenosine deaminase, adenosine kinase, and AMP 5'-nucleotidase. In whole brains in vivo, maturation (from 18 days of gestation to 14 days post partum) was associated with an increase in the activities of guanase, PNP, IMP 5'-nucleotidase, AMP deaminase, and HGPRT, a decrease in the activities of adenosine deaminase and IMP dehydrogenase, and no change in the activities of APRT, AMP 5'-nucleotidase, and adenosine kinase. The profound changes in purine metabolism, which occur with maturation of the neuronal cells in primary cultures in vitro and in whole brains in vivo, create an advantage for AMP degradation by deamination, rather than by dephosphorylation, and for guanine degradation to xanthine over its reutilization for synthesis of GMP. The physiological meaning of the maturational increase in these two ammonia-producing enzymes in the brain is not yet clear. The striking similarity in the alterations of enzyme activities in the two systems indicates that the primary culture system may serve as an appropriate model for the study of purine metabolism in brain.     

Factors affecting the onset of deoxyribonucleic acid synthesis during wheat embryo germination. Study of the changes in DNA polymerases A, B and C and the pool of DNA precursors.

DNA synthesis starts about 12 h after water imbibition in wheat embryos. We have determined that noticeable amounts of labelled thymidine are found inside the embryo only after 6 hr of germination. DNA polymerase C from ungerminated wheat embryos decreased markedly in activity during the first hours of germination, whereas the activities of DNA polymerases A and B increased, having a maximum at about 15 h or germination. Serological evidence has suggested a clear antigenic relationship between DNA polymerases A and C. Although the pool of ATP increases rapidly after water imbibition, the increase in the pool of dNTP species was much slower.     

Purine and pyrimidine metabolism during the partial drying treatment of white spruce (Picea glauca) somatic embryos

The imposition of a partial drying treatment (PDT) on mature white spruce somatic embryos is a necessary step for successful germination and embryo conversion into plantlets. Purine and pyrimidine metabolism was investigated during the PDT of white spruce somatic embryos by following the metabolic fate of ¹⁴C-labeled adenine, adenosine, and inosine, as purine intermediates, and orotic acid, uridine, and uracil, as pyrimidine intermediates, as well as examining the activities of key enzymes. Both the salvage and the degradation pathways of purines were operative in partially dried embryos. Adenine and adenosine were extensively salvaged by the enzymes adenine phosphoribosyltransferase and adenosine kinase, respectively. The activity of the former enzyme increased during the PDT. In both mature and partially dried embryos, a large proportion of inosine was recovered as degradation products. The de novo pathway of pyrimidine nucleotide biosynthesis, estimated by the incorporation of orotic acid into the nucleotides and nucleic acids, was high at the end of the maturation period and declined during the PDT. Uridine was the main substrate for the pyrimidine salvage pathway, since a large proportion of uracil was recovered as degradation products, i.e. CO₂ and β - ureidopropionic acid in both mature and partially dried embryos. Uridine was mainly salvaged by uridine kinase, whose activity was found to increase during the PDT. Taken together these results indicate that the PDT might be required for increasing the activity of adenine and uridine salvage enzymes, which could contribute to the enlargement of the nucleotide pool required at the onset of germination.     

Brassinolide-improved development of <Emphasis Type="Italic">Brassica napus</Emphasis> microspore-derived embryos is associated with increased activities of purine and pyrimidine salvage pathways

Cellular brassinolide (BL) levels regulate the development of Brassica napus microspore-derived embryos (MDEs). Synthesis and degradation of nucleotides were measured on developing MDEs treated with BL or brassinazole (BrZ), a biosynthetic inhibitor of BL. Purine metabolism was investigated by following the metabolic fate of ¹⁴C-labelled adenine and adenosine, substrates of the salvage pathway, and inosine, an intermediate of both salvage and degradation pathways. For pyrimidine, orotic acid, uridine and uracil were employed as markers for the de novo (orotic acid), salvage (uridine and uracil), and degradation (uracil) pathways. Our results indicate that utilization of adenine, adenosine, and uridine for nucleotides and nucleic acids increased significantly in BL-treated embryos at day 15 and remained high throughout the culture period. These metabolic changes were ascribed to the activities of the respective salvage enzymes: adenine phosphoribosyltransferase (EC 2.4.2.7), adenosine kinase (EC 2.7.1.20), and uridine kinase (EC 2.7.1.48), which were induced by BL applications. The BL promotion of salvage synthesis was accompanied by a reduction in the activities of the degradation pathways, suggesting the presence of competitive anabolic and catabolic mechanisms utilizing the labelled precursors. In BrZ-treated embryos, with depleted BL levels, the salvage activity of both purine and pyrimidine nucleotides was reduced and this was associated to structural abnormalities and poor embryonic performance. In these embryos, the activities of major salvage enzymes were consistently lower to those measured in their control (untreated) counterparts.     

Evidence for non-equilibrating pools of apolipoprotein C-III in plasma lipoproteins

Using immunoaffinity chromatography to isolate apoC-III from radiolabeled lipoproteins for direct determination of specific radioactivity, we have studied the metabolism of human apoC-III in VLDL and in HDL following the bolus injection of 125I-labeled VLDL. Transfer of apoC-III radioactivity from VLDL to HDL was detected in the plasma sample drawn 5 min after injection of the tracer. However, the specific radioactivity of apoC-III in VLDL was found to be higher than that in HDL, with this difference being maintained throughout the sampling period (48-72 hr). The ratios of the respective specific activities ranged from 1.2 to 1.9 in six subjects studied (two normolipidemics and four hypertriglyceridemics). When 125I-labeled HDL was injected as the tracer, however, the higher apoC-III specific radioactivity was associated with the HDL fraction. This lack of complete equilibration of apoC-III between VLDL and HDL in vivo was further characterized by in vitro studies using either 125I-labeled VLDL or 125I-labeled HDL. All incubations were carried out for 3 hr at 37 degrees C followed by 16 hr at 4 degrees C and the apoC-III specific activity in each lipoprotein fraction was directly determined after immunoaffinity chromatography. In a study of plasma from a mildly hypertriglyceridemic subject in which 125I-labeled VLDL was incubated with unlabeled HDL, apoC-III specific activities in VLDL remained 30% greater than that in HDL. When 125I-labeled HDL (from the same subject) was incubated with unlabeled VLDL of apoC-III, final specific activity in VLDL was less than 10% of that of HDL apoC-III. Differences in specific activities were also demonstrated when radiolabeled purified apoC-III was exchanged onto VLDL prior to its incubation with HDL. A consistent difference in apoC-III specific activities in VLDL and HDL was observed after isolation of the particles either by molecular sieve chromatography or by ultracentrifugation. These studies demonstrated that, while the exchange of apoC-III between VLDL and HDL may be very rapid, this equilibration is not complete. Pools of apoC-III that do not participate in the equilibration process are present in both the VLDL and HDL fractions and could account for 30-60% of the total apoC-III mass in each lipoprotein fraction.     

Synthesis of ribonucleic acid in normal bone in vitro

1. The incorporation of [2-(14)C]uridine into nucleic acids of bone cells was studied in rat and pig trabecular-bone fragments surviving in vitro. 2. The rapid uptake of uridine into trichloroacetic acid-soluble material, and its subsequent incorporation into a crude nucleic acid fraction of bone or purified RNA extracted from isolated bone cells, was proportional to uridine concentration in the incubation medium over a range 0.5-20.0mum. 3. During continued exposure to radioactive uridine, bulk RNA became labelled in a curvilinear fashion. Radioactivity rapidly entered nuclear RNA, which approached its maximum specific activity by 2hr. of incubation; cytoplasmic RNA, and particularly microsomal RNA, was more slowly labelled. The kinetics of labelling and rapid decline of the nuclear/microsomal specific activity ratio were consistent with a precursor-product relationship. 4. Bulk RNA preparations were resolved by zonal centrifugation in sucrose density gradients into components with approximate sedimentation coefficients 28s, 18s and 4s. 5. Rapidly labelled RNA, predominantly nuclear in location, demonstrated a polydisperse sedimentation pattern that did not conform to the major types of stable cellular RNA. Material of highest specific activity, sedimenting in the 4-18s region and insoluble in 10% (w/v) sodium chloride, rapidly achieved its maximum activity during continued exposure to radioactive precursor and decayed equally rapidly during ;chase' incubation, exhibiting an average half-life of 4.3hr. 6. Ribosomal 28s and 18s RNA were of lower specific activity, which increased linearly for at least 6hr. in the continued presence of radioactive uridine. There was persistent but variable incorporation into ribosomal RNA during ;chase' incubation despite rapid decline in total radioactivity of the acid-soluble pool containing RNA precursors.     

Role of adenosine salvage in wound-induced adenylate biosynthesis in potato tuber slices.

Levels of ATP and other nucleotides increased in wounded potato tuber slices, maintained on moist paper for 24 h after preparation. The relative expression intensity of genes encoding adenosine kinase (AK) and adenine phosphoribosyltransferase (APRT) in wounded slices was greater than the intensity of genes of the de novo pathway, glycineamide ribonucleotide formyltransferase (GART) and 5-aminoimidazole ribonucleotide synthetase (AIRS). In vitro activities of adenosine kinase (ATP:adenosine 5'-phosphotransferase; EC 2.7.1.20) and adenine phosphoribosyltransferase (AMP:pyrophosphate phospho-d-ribosyltransferase; EC 2.4.2.7) increased during wounding. Adenosine nucleosidase (adenosine ribohydrolase; EC 3.2.2.7) activity was negligible in freshly prepared slices, but its activity is dramatically enhanced in wounded slices. In situ adenosine salvage activity, estimated from the incorporation of radioactivity from exogenously supplied [8-(14)C]adenosine into nucleotides and RNA, increased more than five times in the wounded slices. These results strongly suggest that greater expression of the genes encoding enzymes of adenosine salvage during wounding is closely related to the increased supply of adenine nucleotides in the wounded slices.     

Adenine nucleotide metabolism in relation to purine enzymes in liver,erythrocytes and cultured fibroblasts

To evaluate the regulation of adenine nucleotide metabolism in relation to purine enzyme activities in rat liver, human erythrocytes and cultured human skin fibroblasts, rapid and sensitive assays for the purine enzymes, adenosine deaminase (EC 2.5.4.4), adenosine kinase (EC 2.7.1.20), hypoxanthine phosphoribosyltransferase (EC 2.4.28), adenine phosphoribosyltransferase (EC 2.4.2.7) and 5′-nucleotidase (EC 3.1.3.5) were standardized for these tissues. Adenosine deaminase was assayed by measuring the formation of product, inosine (plus traces of hypoxanthine), isolated chromatographically with 95% recovery of inosine. The other enzymes were assayed by isolating the labelled product or substrate nucleotides as lanthanum salts. Fibroblast enzymes were assayed using thin-layer chromatographic procedures because the high levels of 5′-nucleotidase present in this tissue interferred with the formation of LaCl₃ salts. The lanthanum and the thin-layer chromatographic methods agreed with-in 10%.Liver cell sap had the highest activities of all purine enzymes except for 5′-nucleotidase and adenosine deaminase which were highest in fibroblasts. Erythrocytes had lowest activities of all except for hypoxanthine phosphoribosyltransferase which was intermediate between the liver and fibroblasts. Erythrocytes were devoid of 5′-nucleotidase activity. Hepatic adenosine kinase activity was thought to control the rate of loss of adenine nucleotides in the tissue.Erythrocytes had excellent purine salvage capacity, but due to the relatively low activity of adenosine deaminase, deamination might be rate limiting in the formation of guanine nucleotides. Fibroblasts, with high levels of 5′-nucleotidase, have the potential to catabolize adenine nucleotides beyond the control of adenosine kinase. The purine salvage capacity in the three tissues was erythrocyte > liver > fibroblasts. Based on purine enzyme activities, erythrocytes offer a unique system to study adenine salvage; fibroblasts to study adenine degradation; and liver to study both salvage and degradation.     

De Novo Biosynthesis of Deoxyribonucleic Acid Polymerase during Wheat Embryo Germination

An 8-fold enhancement in the activity of a DNA-dependent DNA polymerase was found in extracts from germinating wheat (Triticum vulgare var. Florence) embryos, as compared to the activity found in extracts from ungerminated embryos. The enhancement of this activity during the first hours of germination is concomitant to the increase of a Dnase activity. The two activities could be separated and the increased level of the DNA polymerase upon germination was observed in an enzymatic fraction which contains very low DNase activity. Addition of the protein synthesis inhibitor, blasticidin S, to germinating wheat embryos, reduced the increase in DNA polymerase. Incorporation of radioactive amino acids into a phosphocellulose preparation, which contains the DNA polymerase starts during the first 6 hours of germination. The amount of radioactivity incorporated is doubled in the next 6 hours, and the incorporation is continued between 12 and 18 hours of germination.     

Biological activity, binding, and metabolic fate of Ac-[Nle4, D-Phe7]alpha-MSH4-11NH2 with the F1 variant of B16 melanoma cells

The alpha-MSH (alpha-melanocyte-stimulating hormone) agonist, Ac-[Nle4, D-Phe7]alpha-MSH4-11NH2 (hereafter called ND4-11 alpha-MSH), is at least 10-fold more potent than alpha-MSH as a stimulus of tyrosinase activity in F1 variant cells of B16 melanoma. The binding to these cells during an incubation with 5 nM (3H)ND4-11 alpha-MSH at 37 degrees C is maximal at 0-30 min, 22 fmol/10(6) cells, but declines to 40% of this value at 4 hr. in the presence of 5 nM (3H)ND4-11 alpha-MSH at 37 degrees C, the acid soluble (cell surface) radioactivity decreased rapidly from 11.4 fmol/10(6) cells at 5 min to 4.6 fmol/10(6) cells at 4 hr. Chromatographic analysis of media and cellular samples revealed that there was no evidence of degradation of (3H)ND4-11 alpha-MSH in the medium but there was evidence of intracellular degradation of (3H)ND4-11 alpha-MSH. Ammonium chloride (10mM) resulted in an increase in acid resistant radioactivity (internalized hormone) at 4 hr. The binding to F1 variant cells during an incubation with 0.155 nM or 5 nM (3H)ND4-11 alpha-MSH at 4 degrees C was constant from 4 hr to 24 hr. Under these conditions, there was no time-dependent change in the acid soluble radioactivity from 4 to 24 hr. Scatchard analysis of (3H)ND4-11 alpha-MSH binding to F1 variant cells at 4 degrees C demonstrated that there were approximately 4500 receptors per cell and an association constant of 17.1 nM-1. These results are consistent with a process of (3H)ND4-11 alpha-MSH binding to its receptor followed by internalization of the receptor-hormone complex and then intracellular degradation of the hormone.